HIV-1 antiretrovirals induce oxidant injury and increase intima-media thickness in an atherogenic mouse model

Abstract

A growing body of evidence suggests HIV patients are at a greater risk for developing atherosclerosis. However, clinical investigations have generated conflicting results with regard to whether antiretrovirals are independently involved in the development of HIV-associated atherosclerosis. By administering antiretrovirals in an atherogenic mouse model, we determined whether two commonly prescribed antiretrovirals, the protease inhibitor indinavir and the nucleoside reverse transcriptase inhibitor AZT, can induce premature atherosclerosis. C57BL/6 mice were administered an atherogenic diet+/-AZT, indinavir, or AZT plus indinavir for 20 weeks. Aortic intima-media thickness (IMT) and cross-sectional area (CSA) were determined. Compared to controls, treatment with AZT, indinavir or AZT plus indinavir, significantly increased aortic IMT and CSA. This suggests that antiretrovirals can directly exacerbate atherogenesis, in the absence of interaction with a retroviral infection. To elucidate the role of oxidant injury in the drug-induced initiation of atherosclerosis, a separate group of mice were treated for 2 weeks with an atherogenic diet+/-AZT, indinavir or AZT plus indinavir. Aortic reactive oxygen species (ROS) production and glutathione/glutathione disulfide (GSH/GSSG) ratios, as well as plasma levels of 8-isoprostanes (8-iso-PGF(2alpha)) and lipids were determined. At 2 weeks, aortic ROS was increased and GSH/GSSG ratios were decreased in all antiretroviral treatment groups. Plasma 8-iso-PGF(2alpha) was increased in the AZT and AZT plus indinavir-treated groups. At 20 weeks, increased ROS production was maintained for the AZT and indinavir treatment groups, and increased 8-iso-PGF(2alpha) levels remained elevated in the AZT treatment group. Cholesterol levels were moderately elevated in the AZT and AZT plus indinavir-treated groups at 2 but not 20 weeks. Conversely, indinavir treatment increased plasma cholesterol at 20 but not 2 weeks. Thus, though effects on plasma lipid levels occurred, with effects of the individual antiretrovirals variable across the treatment period, there was consistent evidence of oxidant injury across both early and late time points. Together with the known metabolic abnormalities induced by antiretrovirals, drug-induced oxidant production may contribute to the development of antiretroviral-associated atherosclerosis.

Figure 1

Intima-media thickness (A) and intima-media cross sectional area (CSA; B) in aortas of C57BL/6 mice fed a high fat, high cholesterol diet ± antiretrovirals for 20 weeks. Values are means ± standard error. * Indicates significant differences in the antiretroviral-treated mice as compared to controls, as revealed by one-way ANOVA with Tukey posthoc t-tests.

Figure 2

Vascular reactive oxygen species (ROS) measured by lucigenin-enhanced chemiluminescence in aortic sections from mice treated for 20 weeks with an atherogenic diet ± antiretrovirals. Aortas were cut into 3 mm length rings, were added to a microplate containing 5 μM lucigenin in 2 mL Kreb’s solutions and chemiluminescence was detected for 10 min. AZT and indinavir treatments significantly increased vascular superoxide generation in aortic rings. * Indicates statistical significance versus controls (p< 0.05).

Figure 3

Plasma 8-iso-PGF_2α levels in mice fed an atherogenic diet ± antiretrovirals for 20 weeks. The 8-isoprostane levels were assessed by ELISA following solid-phase extraction. * Indicates statistical significance versus controls (p<0.05).

Figure 4

Aortic reactive oxygen species production measured at 2 weeks after treatment with a high fat diet A) or a high fat diet plus B) AZT, C) indinavir, or D) AZT plus indinavir. Aortic cross-sections taken near the origin were incubated with 2μM dihydroethidium. Total magnification for all images is approximately 200x. The arrows indicate vascular endothelium, “M” denotes the medial layer, and “L” indicates the location of the vessel lumen. E) Quantitation of reactive oxygen species in aortic cross sections of mice treated for 2 weeks with a high fat diet ± antiretrovirals. DHE-stained images were analyzed for color density using Image J software. Data represent mean densities ± standard error. ANOVA revealed a significant effect of treatment (* P<0.05 versus controls).

Figure 5

Glutathione-to-glutathione disulfide (GSH/GSSG) ratios for aortic tissues of mice treated for 2 weeks with a high fat diet ± antiretrovirals. Data represents means ± standard error. ANOVA revealed a significant effect of treatment compared to controls (* P<0.05).